Immuno assays are used in a wide variety of applications in the field of medicine. Such assays are tests to determine the concentrations of various substances present in blood samples taken from a patient. There are many known assays for particular substances. One of the more common classes of assays is to determine the concentration of particular hormones in a patient's blood. For example, thyroxin, also called T4, is a hormone which regulates human metabolism. It is desirable to screen all newborn babies for a shortage of T4 since such a condition can cause irreversible mental retardation if not treated. Similarly, assays for triiodothyroxine also known as T3 may be used to detect hyperthyroid conditions in patients.
Assays also exist for the presence of viruses and bacteria in a patient. Also, some modern drugs which are used to treat patients become toxic if allowed to accumulate in high concentrations in the patient's blood and, ultimately, do more harm than good. One example is the drug digoxin which is used to regulate the heart beat in cardiac patients. The problem of treatment with digoxin is that there is a wide variation from patient to patient in the amount of the drug required to produce particular concentrations in the bloodstream and excess concentrations of digoxin are toxic.
Modern immuno assay systems fall generally into three categories: (1) radio immuno assays; (2) enzyme immunoassays; and (3) fluoro immuno assays. The common feature of each is the use of marked or labeled standard solutions. A typical immuno assay scheme involves the preparation of an antibody specific to the substance (antigen) for which the test is to be conducted. Samples of the antigen are prepared by the tester which have the marker in question attached to each molecule. In the case of radio immuno assays (RIA) the labeling agent is a radioactive substance. In the case of fluoro immuno assays (FIA), the labeling agent is a material having known fluorescent qualities. Because Enzyme immunoassays are measured spectrophotometrically, they are not as sensitive as radioimmunoassays or flouro immuno assays.
The next step is to provide a dose response curve which is arrived at by mixing the labeled or antigen solution with differing concentrations of a standard unmarked antigen solution and allowing these various mixtures to compete for antibody sites in a container such as a microtiter well which has been coated with a specific antibody. The marked and unmarked antigens in the mixtures compete for the antibody sites and become attached to the antibody sites in proportion to the presence in their mixture.
The remainder of the mixture is removed from the well containing the antibodies by aspiration or some other method and then tested for the presence of the labeled antigens. Since the labeled solution was mixed with various known concentrations of unlabeled antigen, a dose response curve may be drawn, either by hand or with the aid of a computer, to correlate the output of the label detector to the concentration of the unlabeled antigens for the particular sample of marked antigen solution being used. Thus, the dose response curve provides a way of directly translating the amount of marked antigen attached to the antibody sites to the concentration of the unmarked antigen with which the marked solution was mixed.
When this has been accomplished, samples of the marked solution will be mixed with samples of patient's serum, and the marked antigen molecules will compete with the patient antigen molecules for the antibody sites. In the same fashion recited above, the patient antigen molecules and the labeled antigen molecules will compete for the available antibody sites and will be successful in proportion to their relative concentrations in the solution.
When the remainder of the solution is removed, the container is tested for the presence of the label. By using the dose response curve (which is for the particular marked solution being used) the amount of the labeled antigen which remains attached to the antibody sites will provide a direct indication off the dose response curve of the concentration of the particular antigen in the patient serum sample.
In the case of RIA, the most common label used is a radioactive isotope of iodine, .sup.125 I. In testing for the amount of labeled antigen attached to the antibody sites, the radioactive emissions from the samples must be counted in order to get a reading off the dose response curve indicative of the concentration of the antigen in the patient serum.
Radio immuno assays have become very popular in that they provide assays of very good reliability and sensitivity. The fundamental drawback of radio immuno assays is that they are quite expensive. First, the apparatus required to test for the presence of the radioactivity is complex and expensive. Secondly, the particular isotope of iodine used has a radioactive half life of sixty days. Solutions of antigens labeled with radioactive iodine must be used very shortly after preparation since their shelf life is severely limited by the rapid radioactive decay of the marker substance. It will thus be appreciated that the economics of distribution are such that only small amounts are provided to each user at any one time and must be shipped very rapidly from point of preparation to point of end use.
Also, there has been a growing reluctance on the part of many shippers, particularly airlines to transport radioactive materials.
In order to overcome these basic drawbacks of RIA techniques, fluoro immuno assays have been created. The main advantage of fluoro immuno assays over radio immuno assays is that the fluorescent compounds used as labels to be attached to antigens are very stable relative to the short radioactive half life of markers used in RIAs. Secondly, the expense of the testing apparatus for FIAs is usually less than that for RIAs and the external problems of handling radioactive materials do not arise.
The basic principle of using fluorescent labels in fluoro immuno assays is that certain materials, when illuminated by radiation in the spectrum around visible light, will emit radiation of a lower frequency (longer wavelength) in response to being so illuminated. For most fluorescent materials used in FIAs, the emitted radiation is in the spectrum of visible light, and thus detectors for detecting light may be used to ascertain the presence of the fluorescent label.
It is known that for such fluorescent materials, the frequency of the fluorescent emission is lower than the frequency of the radiation which causes the material to fluoresce. It therefore follows that the wavelength of the fluorescent emission is longer than the wavelength of the radiation illuminating the material. The difference between the wavelength of fluorescent emission and the wavelength of illumination (excitation) is referred to as the Stokes' shift. Each of the materials used as a fluorescent label has particular characteristics of required wavelength of excitation and resulting wavelength of fluorescent emission, and thus has a characteristic Stokes' shift.
There are two major drawbacks to prior art fluoro immuno assays: (1) the fluorescent marker materials used have been characterized by a relatively low Stokes' shift on the order of twenty to thirty-five nanometers; and (2) the wavelengths of fluorescent emission for the fluorescent markers are very close to the wavelengths of auto fluorescence exhibited by components which are often present in the patient's serum.
The first drawback mentioned above is one which requires very sensitive detectors and complex optical apparatus to distinguish between light having a wavelength characteristic of the fluorescent material and the light used to excite the fluorescent material. Because of the low Stokes shift, it is difficult and expensive to design light sensors which will respond to the wavelength of fluorescence and be relatively insensitive to the wavelength of the excitation light. In order to overcome this problem, many prior art fluoro immuno assay devices have used expensive defraction gratings inserted between the sample containing the fluorescent labels and the optical sensor. These are placed so that they are orthogonal to the direction of a beam of light at the excitation wavelength.
The second drawback noted above reduces the sensitivity and reliability of prior art fluoro immuno assays relative to RIAs. The presence of autofluorescing components in the serum require extra precautions and extra steps to assure that these autofluorescing substances are removed from the sample containing the antibodies before the ultimate test for presence of fluorescent material is made. This increases the complexity and expense of preparing the samples. Furthermore, it is difficult to assure removal of all of the autofluorescing substances and thus the reliability of previous fluoro immuno assays has tended to be less than that of radio immuno assays.